Macromomycin (MCR), an antibiotic protein (molecular weight approximately 15000) from Streptomyces macromomyceticus, is active against tumors in experimental animals with L1210 and P388 leukemias, B16 melanoma, and Lewis-lung sarcoma. MCR is cytotoxic to mammalian cells and studies on the mechanism of action suggest that MCR binds to the mammalian cell surface resulting in the inhibition of DNA synthesis. The mechanism of action of MCR is very similar to that of neocarzinostatin (NCS), another antitumor protein, which according to our studies is also cytotoxic by its action at the cell surface. Thus, MCR and NCS appear to belong to a new class of antitumor drugs with a unique mechanism of action. MCR preparations, obtained commercially or through the National Cancer Institute, are highly impure and unstable. A prerequisite for any pharmacology or clinical study is the availability of a highly purified drug. Therefore, it is proposed to purify MCR to chemical and physical homogeneity and to study the purified preparation for the following: a) stability of the protein in aqueous and nonaqueous solvents, b) development of a reproducible in vitro assay procedure in leukemic cells, c) establish antitumor activity in experimental animals, d) primary structure analysis, e) preparation of a biologically active and radiolabeled MCR, f) development of a sensitive radioimmunoassay procedure, and g) elucidate the mode of action of MCR in leukemic cells. The data obtained from these studies should be useful in experimental pharmacology studies and in future clinical use.